Hemagglutinin TMD peptide inserts into membrane bilayer at acidic and neutral pH. (A) The blue shift and enhancement of fluorescence intensity of tryptophan residues in TMD when incubated in DMPC:DMPG vesicles attest to the location of TMD in the membrane hydrophobic milieu. The emission maximum for tryptophan in an aqueous environment is 350 nm. (B) KSV acrylamide quenching measurements also indicate deep insertion of TMD into the membrane interior. The dramatic decrease in KSV in the vesicular dispersion compared with that in PB buffer shows that tryptophan side chains are embedded deep into the membrane. Moreover, a twofold reduction in KSV, as well as decreased KSV on neutralization, upon incubating in PC:PG vesicles at pH 5.0 compared with that at pH 7.4 suggests that the TMD penetration is deeper at acidic pH.

Self-assembly of TMD in the membrane bilayer can be deduced from Rhodamine self-quenching by variation of composition of the fluorescent-labeled peptide

Rhodamine composition experiments detect tight self-association of TMD and non-random interaction of TMD:FP association. (A) The large self-quenching (i.e. low intensity) of Rhodamine is virtually unchanged in the x = 0.3–1.0 region as the labeled TMD manifests packing of TMD molecules into a tight subunit in the membrane at pH 5.0 and 7.4. In contrast, labeled FP exhibits less self-quenching, indicative of a loose association for the peptide molecules. (B) Association between TMD and FP in the bilayer is not arbitrary as FP of HIV-1 gp41 causes no change in Rho-TMD dequenching or Rho-FP of gp41 dequenching was not affected by mixing with TMD. Change in Rho-FP or Rho-TMD of HA2, in contrast, is obvious when complexed to their counterpart. Note that the smallest value of x in the measurements is 0.02 for (A) and 0.05 for (B). (C) A higher propensity of self-association for TMD than FP is revealed by SDS-PAGE. Lanes 1 and 2 show that FP has less tendency than TMD to form oligomers in SDS in either neutral or acidic buffer. In contrast, TMD formed multiple oligomeric species (lane 4) at pH 4.8 for which minimal association owing to disulfide linkage is expected. The association between TMD and FP is not strong enough to sustain the dispersing force of SDS detergent and the electric field as seen in lane 3.

Assembly of TMD and TMD:FP complex of HA as probed by the Rhodamine self-quenching. The FP peptide of HIV gp41 was used as a negative control. Values are expressed as a percentage of the Rhodamine intensity in the presence of 0.2% Triton X-100.

It is noteworthy that a substantial Rho-TMD dequenching upon addition of FP is observed in the composition variation study, particularly at low x values (Figure 2B) while little dequenching is found for Rho-TMD complexed to FP. Conceivably, the long-range interaction between the fluorescentlabels attached to TMD, which is monitored in the low x regime (Figure 2B), is affected by FP addition; however, the short-range interaction probed by experiments leading to data in Table 1 using fully labeled TMD exhibits little change with FP addition, indicating a very compact TMD oligomer (possiblytrimer) subunit un-dissociable by complexing to FP.

Again the association for both FP and TMD in the complex is tighter at acidic pH than neutral pH (Table 1).

FRET measurements between NBD and Rhodamine afford evidence for interaction between TMD and FP

NBD-Rho FRET efficiency as a function of acceptor concentration. NBD (donor) and Rhodamine (acceptor) were labeled at the ends of FP and TMD peptides, respectively, to examine interaction between the two molecules. Different combinations are depicted by various curves as indicated and the dashed curve is derived from random distribution of R0 = 60 Å donor-acceptor pair [36]. Higher FRET efficiency from experimental data for the labeled NBD-Rho pair than that from the theoretical computation at any given Rhodamine concentration suggests association between TMD and FP in the membrane bilayer.

FP molecules are arranged in antiparallel orientation in the TMD:FP complex

FRET measurements disclose interaction between TMD and FP in an antiparallel manner. The efficiency of FRET between pyrene and NBD labeled to the N- and C-termini of TMD and FP peptides in different combinations is compared to determine the orientation of the TMD:FP complex. FRET efficiency is larger for the donor and acceptor fluorophores attached to the opposite ends of TMD and FP. It is also noted that the interaction between FP and TMD is stronger at pH 5.0 than at 7.4 as reflected by greater transfer efficiency.

Insertion depth of HA2 FP is altered by the interaction with TMD

It has been shown by Tatulian and Tamm [10] that TMD inserted into the membrane nearly perpendicular to the membrane surface. On the other hand, HA2 FP has been found to insertobliquely into the membrane. Hence, it is of interest to examine the effect of TMD:FP formation on the membrane insertion depth and angle of FP. As illustrated in Figure 5, KSV of cobalt quenching of NBD labeled at the N-terminus of FP decreases with the introduction of TMD. In stark contrast, KSV increases upon complexing to TMD for NBD labeled at the C-terminus of FP. The effect of adding TMD on KSV is the same for pH 5.0 and 7.4. The data strongly suggest that the N-terminalportion of FP penetrates deeper while the C-terminus shallower as the TMD:FP complex forms in the membrane. Importantly, as discussed in the following, the alteration of the insertion depth of the N- and C-termini of FP upon complex formation leads to the idea that FP aligned more parallel to TMD with its N-terminus close to the C-terminus of TMD. The finding may have a bearing on the role of TMD in promoting membrane hemifusion to complete fusion transition, as is elaborated in the Discussion. Compatible with the previous results [18], insertion of FP is deeper at acidic pH.

Figure 5

FP inserts deeper into the membrane on association with TMD as probed by KSV values measured from NBD quenched by Co2+. The increased KSV values of NBD labeled to the C-terminus of FP and the decrease in KSV for NBD N-terminally labeled to FP when interacting with TMD can be rationalized by a better alignment of FP on complexing to TMD. The results also support the notion of FP-TMD interaction in the membrane.

The Tb

Figure 6demonstrates the lack of membrane leakage activity of TMD in comparison with FP. Thus, for TMD in POPC vesicular suspension at pH 7.4, little leakage of encapsulated Tb3+ is observed and the extent of leakage is insignificantly different for TMD:FP complex and FP, indicative of low leakage activity for TMD and no enhancement of the activity of FP when complexed to TMD. It is of interest to note that TMD:FP or FP molecules are able to disrupt the membrane at neutral pH, implying that the pH-dependence of the influenza HA2 resides mainly at or prior to the stage of helix hairpin formation [19].

Figure 6

Demonstration of the lack of membrane leakage activity of TMD in comparison with FP. (A) Membrane leakage experiments using Tb3+/DPA assay to monitor membrane activity of TMD, FP and TMD:FP complex. Both FP and FP:TMD display dose-dependent leakage activity whereas TMD alone exhibits little activity. It is noted that the characteristic time of leakage is approximately 200 s for P/L = 0.05. (B) Profile of the steady-state leakage versus P/L for FP, TMD and FP:TMD. (P/L is the peptide to lipid ratio.)

HA2 TMD inserts into membrane nearly perpendicularly and promotes dehydration but causes less membrane perturbation than FP as revealed by ATR-FTIR measurements

To examine the membrane interaction of TMD, and membrane perturbation of TMD alone and TMD:FP complex, infrared experiments were carriedout. The secondary structure and orientation of TMD, FP and TMD:FP are summarized in Table 2. Helix accounted for 64% of the secondary structure for TMD, in qualitativeagreement with the values obtained by Tatulian and Tamm [10]. No significant change in helix content was observed for TMD:FP complex, whosehelicity is approximately an average of that of TMD and FP. The insertion angle for TMD was found to be 34° with respect to the normal of the membrane, slightly larger than the value reported by Tatulian and Tamm [10]. Similar to the helix content, the insertion angle of TMD:FP helix is an average of that of TMD and FP. We also note in Figure 7 that the extent of dehydration is greater for FP than TMD. Moreover, the membrane perturbation probed by the change in lipid acylchain orientation caused by FP and by TMD (Table 2) revealed that FP has a greater effect than TMD. The lesser membrane-perturbing effect of TMD than FP seenhere is compatible with the results of leakage experiments (Figure 6). The smaller insertion angle for TMD than that for FP and less dehydration of TMD may be correlated with its smaller perturbation on the membrane acyl chain orientation. Importantly, as shown in the inset of Figure 7, the dehydration caused by TMD:FP is more pronounced than FP and TMD individually, indicating a synergetic membrane-perturbing effect of the formation of TMD:FP complex suggesting a role of TMD and FP association in destabilizing the fusing membranes.

Figure 7

The ATR-FTIR absorption bands of amide carbonyl vibration of DMPC:DMPG lipid alone and in the presence of TMD, FP and their complex. The higher-frequency band is assigned to the non-hydrogen bonded lipid owing to dehydration, while the lower-frequency band is assigned to hydrated hydrogen bonded lipid. It is seen that the percentage of dehydrated bands increases as the two peptides form a complex and FP has a higher dehydration level than TMD.

Table 2

Lipid

FP

TMD

Complex

Secondary structure

α-helix (%)

26 ± 3

64 ± 1

46 ± 7

β-sheet (%)

55 ± 5

20 ± 2

28 ± 1

Unordered (%)

9 ± 3

---

9 ± 8

β-turns (%)

11 ± 3

17 ± 4

17 ± 3

Helix axis orientation

RATR

1.82 ± 0.01

2.46 ± 0.22

2.07 ± 0.08

Θ (°)

60 ± 1

34 ± 1

49 ± 2

Beta-strand orientation

RATR

1.18 ± 0.10

N.A.

1.28 ± 0.20

Φ (°)

56 ± 5

N.A.

52 ± 7

Acyl chain tilt angle

RATR

1.09 ± 0.09

1.78 ± 0.45

1.31 ± 0.04

1.62 ± 0.12

δ (°)

27 ± 4

49 ± 12

36 ± 1

45 ± 4

The secondary structure and orientation of helix, beta sheet and lipid acyl chain of FP, TMD and FP/TMD 1:1 complex in DMPC:DMPG 1:1 vesicular solution with L/P = 50 at pH 5.0. Values were obtained by averaging three or four sets of data.

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Discussion

TMD of HA2 inserts into membrane bilayer with a pH-dependent depth

We have shown that HA2 FP penetrated more deeply into the membrane at low pH. The result in Figure 1 on TMD membrane-insertion depth displays similar pH dependence. The deeper insertion at acidic pH for both TMD and FP, as discussed in the following, may have ramifications for the low-pH activation of HA2-mediated fusion process.

Self-assembly of TMD is stronger than FP and is insignificantly affected by the incorporation of FP

A previous investigation revealed loose self-association of FP in the membrane [16]. Here we show in Table 1 that TMD molecules form tightly packed oligomeric subunits in the membrane which are tighter than FP as deduced from the greater Rhodamine self-quenching for TMD. No discernible dequenching is observed for Rhodamine-labeled TMD as FP is added, while Rhodamine conjugated to FP has enhanced dequenching with TMD incorporation. This suggests that tight TMD packing is intact upon interacting with FP whereas inter-FP distance becomeslonger for loosely aggregated FP monomers when attracted by tightly associated TMD oligomers nearby. Another line of evidence for a more stable oligomer formed by TMD can be visualized in Figure 2C, in which only the monomeric FP band is displayed. More indirect evidence for tighter association of TMD than FP and that TMD constitutes the inner core of the TMD:FP complex can be deduced from Figure 2B. The association between the two kinds of molecules is further affirmed by the FRET results shown in Figure 3 indicating larger transfer efficiency than random distribution of the two peptides from NBD to Rhodamine conjugated, respectively, to TMD and FP at the opposite ends. The orientation between TMD and FP can be resolved by FRET experiments in which pyrene (donor) and NBD (acceptor) were labeled to TMD and FP at either N- or C-terminus (Figure 4). The result clearly showed an antiparallel TMD:FP association.

The membrane perturbing effect of TMD and FP has also been studied by ATR-IR measurements as shown in Figure 7. The larger fraction of carbonyl vibrational peak for the TMD:FP complex than that for either TMD or FP reveals a synergistic membrane-perturbing effect of the TMD:FP complex. As membrane dehydration represents a major barrier to fusion, this result suggests that association of the two HA2 domains, primarily by perturbing the membrane bilayer at the fusing site, promotes membrane merger mediated by the influenza hemagglutinin.

In this work, fluorophotometry, such as FRET and Rhodamine self-quenching, was used to study the association between TMD and FP and membrane organization of TMD. It turns out that this is appropriate because the active distance for these fluorescence measurements is in the range of 10–50 Å, which covers the loose interaction between FP and TMD. The loose TMD:FP complex inferred from the present work is in line with the sodium dodecyl sulfate gel electrophoresis experiment in which the two coincubated peptides exhibited separate TMD and FP bands under the electric field and dispersing force of SDS (Figure 2C).

Biological implication of FP:TMD interaction

As elaborated above, it is possible that the TMD oligomers are surrounded by FP on the external surface or loosely associated FP molecules disperse around TMD homo-oligomers. It has been shown that the polarsegmentimmediately following FP of HIV-1 gp41 is conformationally plastic [12,20] and that the tryptophan-richpre-TM stretchpossesses membrane activity [21]. Given the involvement of TMD in the hemifusion-to-complete fusion transition and the stringent length requirement for this function [7] we propose a working model for the late steps of HA-mediated fusion (Figure 8). At the pre-hairpin stage, FP inserts into the target membrane; trimerization is mainly mediated through self-association of the HR1 region while HR2 domain is somewhatunordered. Perhaps owing to the flexibility and membrane activity of the FP-proximal region and the membrane-perturbing pre-TM region [22], refolding of the pre-hairpin structure occurs when HA is exposed to acidic pH, pulling the two apposing membranes close. In the membrane interior, FP and TMD movetowards each other in antiparallel orientation to form a loose complex, with self-assembled TMD surrounded by FP or interspersed with FP in a somewhat straggling manner, in view of the report that fusion activity is retained with the TMD segment replaced by TMD from other membrane proteins [8,9]. In addition to deepening the FP penetration into the lipid bilayer and further deforming the membrane at the fusing site, the loose interaction between TMD and FP may fosterclustering of neighboring FP and the associated TMD molecules, a necessary step for the fusion pore formation and enlargement. We propose that the latter process constitutes a major step for FP and TMD to exert their function. Concomitantly, HR1 and HR2 of the ectodomain form a helix hairpin bundle in the space between the apposing membranes. The free energy released from the rearrangement and conformational change enables the fusion protein and viral and target membranes to surmount the barrier of membrane dehydration and deformation (destabilization) required for membrane coalescence [23]. In other words, the synergetic membrane-perturbing effect (Figure 7) and the deepening membrane penetration of FP resulting from complexing to TMD (Figure 5), in combination with TMD traversing both leaflets of bilayer, eventuallycause the rupture of the inner leaflets of both attending membranes resulting in full fusion by the cooperative FP:TMD clusterrecruited to the fusion site.

Figure 8

Schematic illustration on the role of FP and TMD in the late stages of HA2-mediated fusion. (1) In the pre-hairpin stage, FP inserts into the target membrane following disengagement of HA1 from HA2. The inner leaflet of the bilayer is minimally disrupted by FP with an oblique insertion angle. Note the loose FP self-assembly and tight self-association of TMD in the membrane. (2) Low pH-induced refolding of HR1 and HR2 regions of the HA2 driven by strong interactions between them. The two apposing membranes are pulled in proximity and bulged-out to facilitate the merge. (3) Driven by the energy liberated by HR1-HR2 association and additional force provided by the polar, conformationally plastic linker segment downstream of FP and the membranetropic pre-TM region, the two fusing membranes undergo dehydration, deformation and coalescence of the outer leaflets, causing hemifusion. In the process, the compact TMD homo-trimer approaches the loose FP aggregate and may be interspersed with FP molecules, gradually forming the TMD-FP complex, which is not specific per se, with TMD in the inner core. Nonetheless, the interaction is sufficiently strong to align FP with TMD to a certain extent and deepen FP penetration into the inner leaflet, further destabilizing the bilayer. (4) Partly as a result of the complex formation-enhanced perturbation of both leaflets of the effector and target membranes, the hemifusion diaphragm transits to an inceptive fusion pore, concomitant with the six-helix bundle formation of HR1 and HR2. By this stage, the recruitment of adjacent TMD:FP triplex subunits cooperatively stabilizes the initial pore and its dilation to facilitate the mixing of cytoplasmic contents.

We have provided severallines of evidence for the loose association between TMD and FP in the model membrane, in contrast to highly specific recognition of the receptor by the surface subunit of the viral fusion protein. Perhaps the role of TMD in the membrane fusion is twofold: first, mechanically it anchors the fusion protein onto the viral membrane and secures the oligomerization of the fusion protein through its tight self-association, and importantly, it does not destabilize the membrane in the absence of FP; second, it has a weak interaction with FP, therebyreinforcing the destabilizing effect of FP on the inner leaflet of the target membrane by deepening FP membrane insertion (Figure 7). This latter effect is manifested by the requirement of TMD length for different phenotypes of fusion, hemifusion and full-fusion activity [7], because spanning both leaflets of the bilayer for TMD is conceivably a prerequisite for TMD to execute this function. The differential results on the effect of altering the basic residue in the middle of the HIV-1 TMD sequence [9,24] may be related to the weak association between TMD and FP deduced herein (Figure 2C). The concept that the role of TMD in the fusion process lies more in disrupting the inner leaflet of the fusing membranes than the specific interaction with FP is consistent with the inability of a GPI-anchored HA ectodomain to mediate full fusion [6].

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Conclusion

The results presented in the work highlight the importance of the interaction of TMD with the membrane and TMD in complex with FP in the steps leading to pore initiation and dilation and shed some light on the fusion reaction mediated by other type I viral fusion proteins.

where F0 is the fluorescence intensity at the zero quencher concentration, F is the fluorescence intensity at any given quencher concentration [Q], whereas KSV represents the apparent Stern-Volmer quenching constant, obtained from the slope of the plot of F0/F versus [Q].

Rho-labeled/unlabeled peptide composition experiments

In the experiments on the composition variation of Rho-labeled peptide, the fraction of labeled peptide, x, was varied from 0.02 or 0.05 to 1. For self-association measurements of HA2 TMD or FP, the concentrations were kept at 1 μM/100 μM/100 μM of peptide/DMPC/DMPG. To investigate the association between TMD and FP of HA2 or HIV, a total concentration of 0.06 μM of each peptide (labeled and unlabeled) in DMPC:DMPG 30 μM:30 μM was used. Excitation and emission wavelengths of 530 and 578 nm, respectively, were used with slit bandwidth of excitation and emission of 10 nm. The normalized emission intensity Ix/x was plottedagainst 1 - x [28].

It is noted that intra-trimeric interaction is detected for x values near 1 since nearly all peptide molecules are labeled and, therefore, quenching arisespredominantly from the close neighbors within the same trimer. In contrast, for low x values, the probability of finding a pair of labeled peptides is slim and hence quenching arises mainly from labeled peptides in nearby trimers.

Association tendency of TMD and FP by Rho fluorophore

The Rho self-quenching experiments were carried out to examine the propensity of association of TMD with FP. To DMPC:DMPG (30/30 μM) vesicles at pH 5.0 or 7.4, the Rho-labeled TMD (or FP) was added followed by adding the unlabeled FP (or TMD). We used 0.06 μM of each peptide and the parameters were the same as those used in the Rho composition experiments described above. The 100% reference intensity was taken from the fluorescence measured in the peptide/lipid dispersion solubilized with 0.2% (v/v) Triton X-100.

FRET between Rho-labeled TMD peptide and NBD-labeled FP

The Förster distance (R0), at which the FRET efficiency is 50%, of the NBD-Rho pair (donor-acceptor) is about 56 Å [29]. NBD and Rho were labeled on FP and TMD peptides, respectively, at either N- or C-terminal end. The FRET between NBD and Rho was measured at 50°C by adding Rho-TMD to NBD-FP/DMPC/DMPG 0.06:150:150 μM. The ratios of [Rho-TMD]/[NBD-FP] were 0.3, 0.6, 1, 1.5, 2 and 2.5. To investigate the changes of NBD intensity, the excitation and the emission wavelengths were set at 467 and 530 nm, respectively, with a response of 0.04 s and slit bandwidth of excitation and emission of 10 nm.

To calculate the FRET efficiency, the intensity of donor (NBD-FP) without acceptor (Rho-TMD) was taken as 100%:

Efficiency (%) = Idonor+acceptor/Idonor × 100

where Idonor+acceptor and Idonor are the intensities of NBD-FP/Rho-TMD mixture and NBD-FP only, respectively.

FRET between Pyrene-labeled TMD peptide and NBD-labeled FP

The measurements of FRET from Pyrene to NBD were recorded to investigate the alignment between TMD and FP peptides. The Förster distance R0 of the pyrene-NBD pair (donor-acceptor) is about 33 Å [29]. TMD and FP peptides were labeled by pyrene and NBD, respectively, on either N-terminus or C-terminus. Pyrene-labeled TMD was added to the DMPC:DMPG (15:15 μM) vesicular solution followed by the addition of the same amount of NBD-labeled FP. The final concentration of each peptide was 0.06 μM. To monitor the pyrene probe, the excitation and the emission wavelengths were set at 344 and 380 nm, respectively, with slit bandwidth of excitation and emission of 10 nm.

FRET efficiency is calculated according to (2) except that Idonor+acceptor and Idonor are the intensities of pyrene-TMD/NBD-FP mixture and pyrene-TMD only, respectively.

Co

NBD fluorescence can be quenched by divalent cobalt ions [30] via a collisional quenching mechanism. Similar to acrylamide quenching of Trp, a large quenching constant by the aqueous cation reflects a closer proximity of NBD tag to the membrane interface. For Co2+ quenching experiments, the fluorescence of NBD-FP with/without TMD peptide in DMPC:DMPG 15:15 μM vesicles at pH 5.0 or 7.4 was measured until the intensity attained a steady value. The final concentration of each peptide was 0.06 μM. An incremental amount of CoCl2 stock solution (0.1 M) was then injected into the cuvette to give final Co2+ concentration in the range 0.04–2.0 mM. Corrections owing to dilution were made to the observed fluorescence intensities. All parameters were the same as those used for NBD-Rho FRET experiments and the data were analyzed using (1).

Tb

The method is based on the enhancement of the lanthanidemetal Tb3+ fluorescence when the aromaticchelator DPA is liganded to the ion. Large unilamellar vesicles (LUVs) of POPC containing Tb3+ were prepared as described previously [19,31,32].

To quantitate the extent of leakage observed in the Tb3+/DPA assay, FP or TMD peptide or TMD:FP 1:1 complex were added to a solution containing 40 μM POPC/Tb3+, 50 μM DPA, 100 mM NaCl, 10 mM Tris at pH 7.4. The fluorescence was recorded at ambienttemperature with excitation and emission wavelengths of 270 and 490 nm, respectively, and 10 nm bandwidth for both excitation and emission. The percentage leakage of Tb3+ was calculated as follows:

Leakage (%) = [(Ft - F0)/(Fmax - F0)] × 100

where Fmax is obtained by adding 0.05% (v/v) Triton X-100 and F0 is equivalent to the values for DMSOcontrols.

SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)

HA2 FP and TMD peptides were dissolved in HFIP and mixed with lysoPC (1-dodecyl-2-hydroxyphosphatidylcholine) in ethanol as described by Tatulian and Tamm [10]. The organicsolvents were removed under a stream of nitrogen followed by high vacuum for 1 h. The dried mixtures were then resuspended in either neutral (43 mM imidazole, 35 mM HEPES, pH 7.3) or acidic buffer (80 mM GABA, 20 mM acetic acid, pH 4.8) and sonicated for 6 min before mixing with the Laemmli buffer (pH 6.8) composed of 62.5 mM Tris-HCl, 25% glceryol, 2% SDS and 0.01% Bromophenol Blue. The concentrations of peptide and lysoPC were around 0.5 and 3 mM, respectively, and the pH of the acidic buffer mixed with Laemmli buffer was raised to about 5.3. For each lane of sampleloading, 5 μl of the peptide/lysoPC was mixed with 10 μl Laemmli buffer, except that in lane 3, 5 μl of each of the TMD and FP in lysoPC were mixed before added to 20 μl Laemmli buffer. The molecular weight of each peptide is indicated in parentheses in Figure 2C. Electrophoresis was conducted at 20 mA constant current for 90 min. The image shows the peptide migration in 18% separating gel with 0.1% SDS (pH 8.8). FP exhibits less tendency than TMD to form oligomers in SDS in either neutral or acidic buffer, as shown in lanes 1 and 2. In contrast, TMD formed multiple oligomeric species (lane 4). The association between TMD and FP is not strongenough to counter the dispersing force of SDS detergent and the electric field gradient as seen in lane 3.

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